JP4698023B2 - Method and apparatus for synthesizing DNA probe array - Google Patents

Method and apparatus for synthesizing DNA probe array Download PDF

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JP4698023B2
JP4698023B2 JP2000532704A JP2000532704A JP4698023B2 JP 4698023 B2 JP4698023 B2 JP 4698023B2 JP 2000532704 A JP2000532704 A JP 2000532704A JP 2000532704 A JP2000532704 A JP 2000532704A JP 4698023 B2 JP4698023 B2 JP 4698023B2
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フランチェスコ セリーナ
マイケル アール サッスマン
フレデリック アール ブラットナー
ガッソン サンギート シング
ローランド グリーン
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ウイスコンシン アラムニ リサーチ ファンデーション
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Abstract

The synthesis of arrays of DNA probe sequences, polypeptides and the like is carried out using a patterning process on an active surface of a substrate (12). An image is projected onto the active surface (15) utilizing an image former (11) that includes a light source that provides light to a micromirror device (35) comprising an array of electronically addressable micromirrors (36). The substrate (12) is activated in a defined pattern and bases are coupled at the activated sites, with further repeats until the elements of a two-dimensional array on the substrate have an appropriate base bound thereto. The micromirror array (35) can be controlled in conjunction with a DNA synthesizer to control the sequencing of images presented by the micromirror array (35) in coordination with the reagents provided to the substrate (12).

Description

【０００１】
（技術分野）
本発明は一般に生物学の分野、特にDNA及び関連ポリマーの分析及び配列決定のための技術及び装置に関する。
【０００２】
（背景技術）
デオキシリボ核酸(DNA)の配列決定は最新生物学の基本的な手段であり、通常種々の方法で、普通にはDNAセグメントを電気泳動により分離する方法により行なわれる。例えば、Current Protocols In Molecular Biology, 1巻, 7章,“DNA Sequecing", 1995を参照のこと。幾つかの重要なゲノムの配列決定は既に完結されており（例えば、酵母、E. coli）、研究が医療上及び農業上重要なその他のゲノム（例えば、ヒト、C.エレガンス、アラビドプシス）の配列決定について進んでいる。医療関係では、多数のヒト個体のゲノムを“再配列決定”してどの遺伝子型がどの病気に関連するのかを測定することが必要であろう。このような配列決定技術はどの遺伝子が活性であり、又どの遺伝子が癌の如き特定の組織中で、又は更に一般には遺伝により影響される疾患を示す個体中で不活性であるのかを測定するのに使用し得る。このような研究の結果は新規薬剤に良好な標的であるタンパク質の同定又は遺伝子治療に有効であり得る適当な遺伝子変化の同定を可能にし得る。その他の用途はDNAをあらゆる土壌又は組織サンプルから単離し、全ての既知の微生物からのリボソームDNA配列からのプローブを使用してサンプル中に存在する微生物を同定することができることが望ましいような土壌生態学又は病理学の如き分野にある。
【０００３】
電気泳動を使用するDNAの通常の配列決定は典型的には労力を要し、しかも時間を浪費する。通常のDNA配列決定の種々の別法が提案されていた。フォトリトグラフィー技術により合成されたオリゴヌクレオチドプローブのアレイを利用する、一つのこのような別のアプローチがPeaseら,“Light-Generated oligonucleotide Arrays for Rapid DNA Sequence Analysis", Proc. Natl. Acad. Sci. USA, 91巻, 5022-5026頁, 1994年５月に記載されている。このアプローチでは、光不安定な保護基で修飾された固体担体の表面がフォトリトグラフィーマスクを通して照明され、照明された領域で反応性ヒドロキシル基を生じる。光不安定基で5'ヒドロキシルの位置で保護された、3'活性化デオキシヌクレオシドがその後に表面に与えられて、その結果、カップリングが光に暴露された部位で起こる。キャッピング、及び酸化後に、支持体がすすがれ、表面が第二のマスクを通して照明されてカップリングのための付加的なヒドロキシル基を露出する。第二の5'保護活性化デオキシヌクレオシド塩基が表面に与えられる。プローブの所望の組が得られるまで、選択的光脱保護及びカップリングサイクルが繰り返されて或るレベルの塩基を構築する。このようなフォトリトグラフィー技術（アレイ中の夫々の部位にあるオリゴヌクレオチドプローブの配列が知られている）を使用してオリゴヌクレオチドプローブの高密度のミニチュア化アレイを生じることが可能であり得る。次いでこれらのプローブが、標的にカップリングされた蛍光マーカーの使用により行なわれる特別なプローブにハイブリダイズした標的の検出及び適当な蛍光走査顕微鏡による検査により、DNAの標的ストランドの相補配列について研究するのに使用し得る。ポリマー半導体フォトレジスト（これらは光不安定5'保護基を使用するのではなく、フォトリトグラフィー技術により選択的にパターン化される）を使用するこの方法の変化がMcGallら,“Light-Directed Synthesis of High-Density Oligonucleotide Arrays Using Semiconductor Photoresists", Proc. Natl. Acad. Sci. USA, 93巻, 13555-13560頁, 1996年11月、及びG.H. McGallら,“The Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates", Journal of the American Chemical Society 119, 22号, 1997, 5081-5090頁に記載されている。
これらのアプローチの両方の欠点は４種の異なるリトグラフィーマスクが夫々のモノマー塩基について必要とされ、こうして必要とされる異なるマスクの合計数が合成されるDNAプローブ配列の長さの４倍であることである。必要とされる多くの精密なフォトリトグラフィーマスクを製造する高コスト、及び暴露毎にマスクの再位置決めに必要とされる多重の処理工程が、比較的高いコスト及び長過ぎる処理時間に寄与する。
【０００４】
（発明の開示）
本発明によれば、DNAプローブ配列、ポリペプチド等のアレイの合成がパターン化方法を使用して迅速かつ有効に行なわれる。その方法は自動化され、コンピュータ制御されて特別な研究に受注生産されたプローブ配列を含むプローブの一次元又は二次元のアレイの加工を可能にし得る。リトグラフィーマスクが必要とされず、こうしてリトグラフィーマスクの製造と関連するかなりのコスト及び時間遅延を排除し、時間を浪費する操作及びプローブアレイの加工方法中の多重マスクのアライメントを回避する。
本発明において、DNA合成リンカーが適用された活性表面を有する支持体が加工されるプローブを支持するのに使用される。支持体の活性表面を活性化して第一レベルの塩基を与えるために、高い精度の二次元の光像が支持体に映写され、支持体活性表面のアレイ中のこれらのピクセルを照明し、これらが活性化されて第一塩基に結合する。光が適用されるアレイ中のピクセルに入射する光がOH基を脱保護し、それらを塩基に結合するのに利用できるようにする。この現像工程後に、適当な塩基を含む液体が支持体の活性表面に与えられて、選ばれた塩基が露出部位に結合する。次いで支持体表面の二次元アレイの要素の全てがそれらに結合された適当な塩基を有するまで、その方法が繰り返されて別の塩基をピクセル位置の異なる組に結合する。支持体に結合された塩基が塩基に結合することができる薬品又は結合された塩基の全てを覆うフォトレジストの一つ以上の層で保護され、次いで新しいアレイパターンが映写され、第一の新しい塩基が添加されるこれらのピクセル中の保護物質を活性化するために支持体に像形成される。次いでこれらのピクセルが露出され、選ばれた塩基を含む溶液がアレイに適用され、その結果、塩基が露出されたピクセル位置で結合する。次いでこの方法が塩基の第二レベルでその他のピクセル位置の全てについて繰り返される。次いでプローブ配列の完全な選ばれた二次元アレイが完成されるまで、記載された方法が塩基の夫々の所望のレベルについて繰り返されてもよい。
【０００５】
像は光を電気的にアドレス可能なミクロミラー（これらの夫々が少なくとも二つの別の位置の一つの間で選択的に傾斜し得る）の二次元アレイを含むミクロミラー装置に与える適当な光源を有する像形成装置を利用して支持体に映写される。夫々のミクロミラーの位置の一つにおいて、ミクロミラーに入射する光源からの光が光学軸から離れて、又支持体から離れて偏向され、又、夫々のミクロミラーの少なくとも二つの位置の第二の位置では、光が光学軸に沿って、かつ支持体に向かって反射される。映写光学装置がミクロミラーから反射された光を受け取り、ミクロミラーを支持体の活性表面に正確に像形成する。視準光学装置が光源からの光をミクロミラーアレイ又はビームスプリッターに直接与えられるビームに平行にするのに使用されてもよく、そのビームスプリッターはビームの一部をミクロミラーアレイに反射し、反射された光をミクロミラーアレイからビームスプリッター中に伝播する。ミクロミラーから直接反射され、又はビームスプリッター中を伝播された光が映写光学レンズに誘導され、これらがミクロミラーアレイを支持体の活性表面に像形成する。ミクロミラーアレイ中の選択的にアドレス可能なミクロミラーはそれらに与えられた光を完全に反射し、又は完全に偏向し得るので、ミクロミラーアレイの像は“オン”ピクセルと“オフ”ピクセルの間の非常に高いコントラストを示す。ミクロミラーは又二つより多い位置に指標とされることができるかもしれず、その場合には付加的な光学装置が用意されて単一ミクロミラーアレイ装置を使用して一つより多い支持体の暴露を可能にし得る。加えて、ミクロミラーはそれらへの損傷を生じないであらゆる波長の光を反射し、紫外線〜近紫外線の範囲の光を含む、短波長の光が光源から利用されることを可能にし得る。
【０００６】
ミクロミラーアレイは適当なピクセルアドレスシグナルをミクロミラーアレイに与えて適当なミクロミラーをそれらの“反射”位置又は“偏向”位置にあるようにするコンピュータの制御下で操作される。プローブに添加される塩基の夫々のレベルにおける夫々の活性化工程に適したミクロミラーアレイパターンはコンピュータコントローラーにプログラムされる。こうして、コンピュータコントローラーが支持体に与えられる試薬と協力してミクロミラーアレイにより与えられる像の配列決定を制御する。
一実施態様において、支持体は透明であってもよく、ミクロミラーアレイの像が活性表面の反対である支持体の表面に映写されることを可能にする。支持体は、アレイの活性表面をシールするエンクロージャーで、フローセル内に取り付けられてもよく、適当な試薬がフローセルを通って適当な順序でアレイの活性表面に流されてアレイ中にプローブを構築することを可能にする。
本発明の更に別の目的、特徴及び利点が添付図面と一緒にされる時に下記の詳細な説明から明らかであろう。
【０００７】
（発明を実施するための最良の形態）
図面を参照して、DNAプローブアレイ合成、ポリペプチド合成等に使用し得る例示の装置が図１中に一般に10で示され、二次元アレイ像形成装置11とアレイ像が像形成装置11により映写される支持体12とを含む。図１に示された形態について、支持体は露出された入口表面14とヌクレオチド配列プローブ16の二次元アレイが加工される反対の活性表面15とを有する。説明の目的のために、支持体12は試薬が入口20及び出口21を通って与えられる体積19を密閉する支持体12に取り付けられたフローセルエンクロージャー18とともに図に示される。しかしながら、支持体12は像形成装置11に面し、かつ光が活性表面に映写されることを可能にする透明ウインドーを備えた反応チャンバーフローセル内に密閉された支持体の活性表面15により本系に利用されてもよい。又、本発明は不透明又は多孔質支持体を使用してもよい。試薬は通常の塩基合成装置（図１に示されていない）から口部20及び21に与えられてもよい。
【０００８】
像形成装置11は光源25（例えば、紫外源又は近紫外源、例えば、水銀アークランプ）、出力ビーム27を光源25から受け取り、所望の波長（例えば、365nmのHgライン）のみを選択的に通す任意のフィルター26、及び平行にされたビーム30を形成するためのコンデンサーレンズ28を含む。光源光をフィルター又は単色にするためのその他の装置、例えば、回折格子、ダイクロミックミラー、及びプリズムが又透過フィルター以外に使用されてもよく、一般に本明細書中“フィルター”と称される。ビーム30はビームスプリッター32に映写され、これがビーム30の一部をビーム33に反射し、これが二次元ミクロミラーアレイ装置35に映写される。ミクロミラーアレイ装置35はアレイ装置35に供給されたコントロールシグナルに夫々応答して少なくとも二つの方向の一つに傾斜する個々のミクロミラー36の二次元アレイを有する。コントロールシグナルはコントロールライン39のコンピュータコントローラー38からミクロミラーアレイ装置35に与えられる。ミクロミラー36は、ミラーの第一の位置で、個々のミクロミラー36に衝突する光33の入射ビームの一部が矢印40により示されるように入射ビーム33の斜めの方向に偏向されるようにつくられる。ミラー36の第二の位置では、このような第二の位置でこのようなミラーに衝突するビーム33からの光が矢印41により示されるようにビーム33に平行に逆反射される。ミラー36の夫々から反射された光が個々のビーム41を構成する。多重ビーム41がビームスプリッター32に入射し、減少された強さでビームスプリッターを通過し、次いで、例えば、レンズ45及び46並びに調節可能なアイリス47を含む映写光学装置44に入射する。映写光学装置44は支持体12の活性表面15で個々のビーム41（及びこれらのビームの間の暗領域）により代表されるようなミクロミラーアレイ35のパターンの像を形成するのに利用できる。出射ビーム41はミクロミラー装置と支持体の間に延びる像形成装置11の主光学軸に沿って誘導される。図１に示された形態の支持体12は透明であり、例えば、ヒュームドシリカもしくはソーダ石灰ガラス又は石英から形成され、その結果、49と標識されたラインにより例示された、その上に映写された光が実質的に減衰又は拡散しないで支持体12を通過する。
【０００９】
好ましいミクロミラーアレイ35はテキサス・インストルメンツ社から市販されているデジタル・ミクロミラー装置(DMD)である。これらの装置はアレイ中のミクロミラーを電気的にアドレスすることにより光のパターン化ビームを形成することができるミクロミラー（これらの夫々が実質的に長さ10〜20μｍの辺を有する正方形である）のアレイを有する。このようなDMD装置は典型的にはビデオ映写に使用され、種々のアレイサイズ、例えば、640x800ミクロミラー要素（512,000ピクセル）、640x480（VGA；307,200ピクセル）、800x600（SVGA；480,000ピクセル）、及び1024x768（786,432ピクセル）で入手し得る。このようなアレイが下記の文献及び特許に説明されている：Larry J. Hornbeck,“Digital Light Processing and MEMs: Reflecting the Digital Display Needs of the Networked Society", SPIE/EOS European Symposium on Lasers, Optics, and Vision for Productivity and Manufacturing I, Besancon, France, June 10-14, 1996、並びに米国特許第5,096,279号、同第5,535,047号、同第5,583,688号及び同第5,600,383号明細書。このような装置のミクロミラー36はミラーそれ自体に損傷を生じないで有効な様式で、紫外線及び近紫外線を含む、通常使用可能な波長の光を反射することができる。ミクロミラーアレイ用のエンクロージャーのウインドーは使用される光の波長について最適化された反射防止被覆物をその上に有することが好ましい。ミラー面当り16ミクロンの典型的なミクロミラー装置寸法及び17ミクロンのアレイ中のピッチを有する、480,000ピクセルをコードする、市販のミクロミラーの600x800アレイの利用は、13,600ミクロンｘ10,200ミクロンの合計のミクロミラーアレイ寸法を与える。リトグラフィーレンズについて典型的かつ容易に達成し得る値である、光学系44中の５の減少係数を使用することにより、支持体12に映写された像の寸法はこうして約２ミクロンの解像度で、約2,220ミクロンｘ2040ミクロンである。更に大きい像が多重平行暴露（フローセル18又は像映写機11を段付きにすることによる）を利用することにより、又は更に大きいミクロミラーアレイを使用することにより支持体12に露出し得る。又、所望により、支持体の像の減少だけでなく、拡大をしないで一対一の像形成を行うことが可能である。
【００１０】
映写光学装置44は通常のデザインのものであってもよい。何とならば、形成される像が比較的大きく、かつ回折限界から良く離れているからである。レンズ45及び46は調節可能なアイリス47を通されたビーム41中の光を支持体の活性表面に集中する。映写光学装置44及びビームスプリッター32は、40と標識されたビーム（例えば、軸から10°それた）により示された、主光学軸（ビーム41が平行である映写光学装置44の中心軸）から離れてミクロミラーアレイにより偏向された光が映写光学装置44の入口ヒトミの外に入るように配置される（典型的には0.5/5=0.1；10°は0.1より実質的に大きい0.17の口径に相当する）。アイリス47は有効な口径数を調節し、かつ望ましくない光（特に軸からそれたビーム40）が支持体に伝播されないことを確実にするのに使用される。0.5ミクロン程度に小さい寸法の解像度がこのような光学系で得られる。製造用途について、ミクロミラーアレイ35は365nmに最適化されたリトグラフィーI-ラインレンズの物体焦点面に配置されることが好ましい。このようなレンズは典型的には0.4〜0.5の口径数(NA)で操作し、大きいフィールド容量を有する。
【００１１】
ミクロミラーアレイ装置35は走査系中で段付きにされるミクロミラーの単一ライン（例えば、１ライン中に2,000のミラー要素を有する）で形成されてもよい。この様式では、像の高さがミクロミラーアレイのラインの長さにより固定されるが、支持体12に映写し得る像の幅は実質的に制限されない。支持体12を有するステージ18を移動することにより、ミラーが支持体の夫々の指標位置でサイクルされて支持体活性表面に像形成される像パターンを夫々の新しいラインに形成し得る。
種々のアプローチが支持体12上のDNAプローブ16の加工に利用されてもよく、ミクロリトグラフィー技術の採用である。“直接光加工アプローチ”において、ガラス支持体12がヌクレオチド塩基を結合することができる薬品の層で被覆される。光が映写系11により適用されて、支持体のOH基を脱保護し、それらを塩基に結合するのに利用できるようにする。現像後に、適当なヌクレオチド塩基が支持体の活性表面に流され、通常のホスホルアミジトDNA合成化学を使用して選ばれた部位に結合する。次いでその方法が繰り返されて、別の塩基を位置の異なる組に結合する。その方法は簡単であり、コンビナトリアルアプローチが使用される場合には、順列の数が指数的に増大する。解像度限界は脱保護メカニズムの線形応答により表される。この方法で得られる解像度の限界のために、フォトレジスト技術をベースとする方法が、例えば、McGallらの上記文献に記載されているように、その代わりに使用されてもよい。間接光加工アプローチでは、適合性の化学が２層レジスト系で存在し、この場合、例えば、ポリイミドの第一層が下にある化学の保護として作用し、一方、上部像形成レジストがエポキシをベースとする系である。像形成工程は両方の方法に共通であり、主たる要件は像形成方法に使用される光の波長がヌクレオチド塩基（これらは280nmで特に感受性である）中の転移（化学変化）を励起しないように充分に長いことである。それ故、300nmより長い波長が使用されるべきである。365nmは水銀のI-ラインであり、これはウエハリトグラフィーに最も普通に使用されるものである。
【００１２】
アレイ合成装置10の別の形態が図２の略図に示される。この配置では、ビームスプリッター32が使用されず、光源25、任意のフィルター26、及びコンデンサーレンズ28が主光学軸に或る角度で（例えば、軸に20°で）取り付けられて光30のビームを或る角度でミクロミラー36のアレイに映写する。ミクロミラー36はミラーの第一の位置で光30をオフ軸ビーム40に、又夫々のミラーの第二の位置で主軸に沿ってビーム41に反射するように配向される。その他の面で、図２のアレイ合成装置は図１の装置と同じである。
【００１３】
図２のオフ軸映写配置を使用する好ましいアレイ合成装置の更に詳細な図が図３に示される。図３の装置では、電力源50（例えば、オリエル68820）から出力を与えられた、光源25（例えば、1,000WのHgアークランプ、オリエル6287、66021）が所望の紫外波長を含む光源として使用される。フィルター系26は、例えば、赤外線を吸収し、280nmから400nmまでの範囲の波長の光を選択的に反射するのに使用されるダイクロイックミラー（例えば、オリエル66226）を含む。脱イオン水が入れられた水冷液体フィルター（例えば、オリエル6127）が残りの赤外線を吸収するのに使用される。着色ガラスフィルター（オリエル59810）又は干渉フィルター（オリエル56531）が夫々50nm又は10nmの50％バンド幅を有するHgランプ25の365nmのラインを選択するのに使用される。F/l２要素ヒューズドシリカコンデンサー（オリエル66024）がコンデンサー28として使用され、二つの平とつレンズ52（メレス・グリオット01LQP033及びメレス・グリオット01LQP023）とともに、コーラー照明系を形成する。この照明系はミクロミラーアレイ装置35の16mm x 12mmの活性領域を含むのに丁度充分に大きい直径で365nmの光のおよそ平行にされた一様なビーム30を生じる。このビーム30が装置の面に垂直から測定して20°の角度で装置35に入射する。ミクロミラーアレイ装置35は最後のフィルターから約700mm離れて配置される。ミクロミラーが第一の位置にある場合、ビーム30中の光が系から下向きに偏向される。例えば、このミクロミラー装置では、ミラーはそれらの第一の位置でミクロミラーの面に垂直に対して-10°の角度にあってもよく、光を光学軸から良く離れて反射する。ミクロミラーが第二の位置で、例えば、ミクロミラーの面に垂直に対して+10°の角度で偏向されるように調節される場合、第二の位置でこのようなミクロミラーから反射された光はビーム41中でミクロミラーアレイの面に垂直に出現する。次いでそれらの第二の位置でミクロミラーから反射された光により形成されたパターンが二つの二重レンズ45及び46並びに調節可能なアパーチュア47を含むテレセントリック像形成系を使用してフローセル18中に密閉されたガラス支持体12の活性表面15に像形成される。二重レンズ45及び46の夫々が湾曲表面がほぼ触れるように一緒にされた一対の平とつレンズ（例えば、メレス・グリオット01LQP033及び01LQP037）を含む。第一の二重レンズは短い焦点長さ（01LQP033）側がミクロミラーアレイ装置35に向くように配向され、又、第二の二重レンズはその長い焦点長さ（01LQP037）側がミクロミラーアレイ装置35に向くように配向される。同じレンズを含む二重レンズが使用されてもよく、その場合いずれかの側がミクロミラーアレイ装置に面していてもよい。又テレセントリックアパーチュアと称される調節可能なアパーチュア47が第一の二重レンズの逆焦点面に配置される。それは光学系の角度受け入れを変化するのに使用される。小さいアパーチュア直径がコントラスト及び解像度を改良するのに相当するが、それに応じて像の強さを低下した。図３に示されるように、必要な薬品を供給された通常のDNA合成装置55が独立の制御のもとに又はコンピュータ38の制御のもとにチューブ20及び21によりフローセル18に連結されて薬品の所望の配列を与え得る。アパーチュア47に典型的な直径は約30nmである。レンズ45及び46中の光の通路を示す光線図がこの型の屈折光学系について図４に示される。物体の中心（ミクロミラー装置面）で、端部で、又中間の位置で生じる光線のファンが示される。光学系はミクロミラーアレイ装置の面の倒立像を形成する。
【００１４】
反射光学装置を使用するアレイ合成装置の別の実施態様が図５に示される。例示の系は、赤外線を吸収し、350nmから450nmまでの範囲の波長の光を選択的に反射するダイクロイックミラー（例えば、オリエル66228）から形成されたフィルター系とともに、光源として1,000WのHgアークランプ25（例えば、オリエル6287、66021）を利用する。F/l２要素ヒューズドシリカコンデンサーレンズ（オリエル66024）が365nmラインを含むが、300nm以下の望ましくない波長を除く光30のおよそ平行にされたビームを生じるのに使用される。コーラー照明系が必要により図５の装置中で又使用されてもよく、一様性及び強さを増大し得る。ビーム30は約16mm x 12mmのミクロミラーの活性領域を有し、かつUV源25のノズルから約210nmに配置されるミクロミラーアレイ装置35に入射し、ビーム30はアレイの面に垂直に対して20°の角度でミクロミラー装置35の平面に衝突する。ミクロミラーの第一の位置で、例えば、アレイの面に対して-10°でミクロミラーから反射された光が系から誘導され、一方、第二の位置、例えば、アレイの面に対して+10°にあるミクロミラーからの光が凹形ミラー60及び凸形ミラー61を含む反射テレセントリック像形成系に向かってビーム41中で誘導される。両方のミラーは球形であり、高い反射率のために増進されたUV被覆物を有することが好ましい。ミラー60及び61からの反射を行った後に、ビーム41は別の平面ミラー63に入射してもよく、これはビームをフローセル18に向かって偏向する。ミクロミラーから反射された光がフローセル18中に密閉されたガラス支持体の活性表面に像形成される。テレセントリックアパーチュア（図５に示されていない）が凸形ミラーの前に置かれてもよい。ビーム41は最初に凹形ミラーに衝突し、次いで凸形ミラーに衝突し、次いで再度凹形ミラーに衝突し、平面ミラー63が必要により光を90°偏向してそれをフローセル18に誘導するのに使用されてもよい。示された系について、凹形ミラー60は152.4mmの直径、及び304.8mm（ES F43561）の球形ミラー表面半径を有してもよく、又凸形ミラーは25mmの直径、及び152.94mm（ES F45625）の球形ミラー表面半径を有していてもよい。理想的には、凹形ミラーの曲率の半径は凸形ミラーのそれの２倍である。このような反射光学系は公知であり、“ミクロアライン”型の系で光学リトグラフィーで通常使用される。例えば、A. Offner,“New Concepts in Projection Mask Aligners", Optical Engineering, 14巻, 130-132頁(1975)、及びR.T. Kerthら,“Excimer Laser Projection Lithography on a Full-Field Scanning Projection Systems", IEEE Electron Device Letters, EDL-7(5)巻, 299-301頁(1986)を参照のこと。
【００１５】
図６は図５の光学系に関する像形成を示す。物体（ミクロミラーアレイ装置）の中央、端部、及び中間の位置で生じる光線のファンが図６に示される。光線は最初に凹形ミラー60から反射し、次いで凸形ミラー61から反射し、次いで再度凹形ミラー60から反射して、ミクロミラーアレイ装置の面の倒立像を形成する。平面ミラー63は図６の線図に含まれていない。テレセントリックアパーチュア（示されていない）が凸形ミラーの前に置かれていてもよい。
屈折光学系又は反射光学系は両方とも“対称”であることが好ましく、抹消によりコマ及び球面収差の如き収差を最小にする。以上の反射系は高い強さを生じる高い口径数を有する。図３及び５のテレセントリック光学系の両方が1:1像形成系である。反射系は色収差を排除し、高い解像度を与えるだけでなく、コンパクトかつ安価であるという潜在的な利点を有する。1:1像形成を行うのに好ましい系は凹形ミラーとレンズ及びプリズムを併有するワイン−ダイソン型の系であろう。それは強さを増強する非常に高い口径数を有する。例えば、F.N. Goodallら,“Excimer Laser Photolitography with 1:1 Wynne-Dyson Optics", SPIE 922巻, Optical/Laser Microlithography, 1988、及びB. Ruffら, “Broadband Deep-UV High NA Photolithography System", SPIE 1088巻, Optical/Laser Microlithography II (1989)を参照のこと。
【００１６】
本発明の装置とともに使用し得る反応チャンバーフローセル18の更に詳細な図が図７及び８に示される。これらの図中の例示のフローセル18は、入口ライン20に連結された入口73と出口ライン21に連結された出口75とを有する、ボルト71により一緒に保持された、アルミニウムハウジング70を含む。図８の断面図に示されるように、ハウジング70はボルト71で支持体の上に一緒に固定されている下部ベース78と上部カバー部分79とを含む。支持体12、例えば、透明ガラススライドが上部プレート79と円筒形ガスケット81（例えば、カル・レッツTMから形成されている）の間に保持され、これが順に非反応性ベースブロック82（例えば、テフロンTM）の上に支持され、入口チャンネル85が入口73から支持体12とベースブロック82（これはガスケットによりシールされる）の間に形成されたシールされた反応チャンバー88に延び、又出口チャンネル89が反応チャンバー88から出口75に延びている。ボルト71はねじ込められてもよく、又ねじ込められていなくてもよく、支持体12をカバー部分とベースの間に取り外し可能に固定して、支持体がフローセルの塩基の最小の変位で交換されることを可能にする。好ましくは、図８に示されるように、ゴムガスケット90がプレート79の底部に取り付けられて周辺領域で支持体に対しかみ合って圧力をガスケット81に対して支持体に適用する。所望により、フローセルは又読み取り中にハイブリダイゼーションチャンバーとして使用されてもよい。
【００１７】
DNAプローブの例示の形成方法が図9-14の略図に関して示される。図９は通常のホスホルアミジト化学を使用してシラン層に被覆された光不安定リンカー分子MENPOC-HEGによる、活性表面15を形成するシラン層95を有する、支持体12の被覆を示す。MENPOC-HEG-CEPは18-O-〔(R,S)-(1-(3,4-(メチレンジオキシ)-6-ニトロフェニル)エトキシ)カルボニル〕-3,6,9,12,15,18-ヘキサオキサオクタデカ-1-イルO'-2-シアノエチル-N,N-ジイソプロピルホスホルアミジトである。シラン層をN-(3-(トリエトキシシリル)-プロピル)-4-ヒドロキシブチルアミドからつくった。図９に示された工程では、支持体が光に露出され、活性遊離OH基が光に露出された領域で露出されるであろう。
図10はMENPOC-HEGリンカーの光脱保護及び光に露出される領域100における遊離OH基の生成を示す。図11はMENPOC-HEGの光脱保護から生成された遊離OH基へのフルオレプライムTMフルオレセインアミジトのカップリングを示す。図12はMENPOC-HEGリンカーの光脱保護から生成された遊離OH基へのDMT-ヌクレオチドのカップリングを示す。図13は光に露出された領域100におけるDMT-ヌクレオチドの酸脱保護の工程を示す。図14はDMT-ヌクレオチド-CEPから合成されたpoly-Tオリゴヌクレオチドとのフルオレセインで標識されたpoly-Aプローブのハイブリダイゼーションを示す。
本発明は例示として本明細書に示された特別な実施態様に限定されないが、特許請求の範囲内に入るような全てのこのような改良形態を包含することが理解される。
【図面の簡単な説明】
【図１】本発明のアレイ合成装置の略図である。
【図２】本発明の別のアレイ合成装置の略図である。
【図３】本発明の一般のテレセントリックアレイ合成装置の更に詳細な略図である。
【図４】図３の装置の屈折光学装置に関する例示の光線図である。
【図５】テレセントリック反射光学装置が利用される本発明のアレイ合成装置の更に別の実施態様の略図である。
【図６】図５の装置の反射光学装置に関する例示の光線図である。
【図７】本発明のアレイ合成装置に利用し得る反応チャンバーフローセルの平面図である。
【図８】図７の線8-8に沿って一般に切断された図７の反応チャンバーフローセルの断面図である。
【図９】光不安定リンカー分子による支持体の被覆を示す略図である。
【図１０】リンカー分子の光脱保護及び遊離OH基の生成を示す略図である。
【図１１】リンカー分子の光脱保護により生成された遊離OH基へのマーカーのカップリングを示す略図である。
【図１２】リンカー分子の光脱保護から生成された遊離OH基へのDMT-ヌクレオチドのカップリングを示す略図である。
【図１３】DMT-ヌクレオチドの酸脱保護を示す略図である。
【図１４】DMT-ヌクレオチド-CEPから合成されたpoly-Tオリゴヌクレオチドとのフルオレセインで標識されたpoly-Aプローブのハイブリダイゼーションを示す略図である。[0001]
(Technical field)
The present invention relates generally to the field of biology, and in particular to techniques and apparatus for the analysis and sequencing of DNA and related polymers.
[0002]
(Background technology)
Sequencing of deoxyribonucleic acid (DNA) is a fundamental means of modern biology and is usually performed by various methods, usually by methods of separating DNA segments by electrophoresis. See, for example, Current Protocols In Molecular Biology, Volume 1, Chapter 7, “DNA Sequecing”, 1995. Sequencing of several important genomes has already been completed (eg yeast, E. coli) and sequences of other genomes where research is of medical and agricultural importance (eg human, C. elegans, arabidopsis) Progress on the decision. In the medical context, it may be necessary to “resequence” the genomes of a large number of human individuals to determine which genotype is associated with which disease. Such sequencing techniques measure which genes are active and which are inactive in specific tissues such as cancer, or more generally in individuals who exhibit a disease affected by inheritance. Can be used to The results of such studies may allow the identification of proteins that are good targets for new drugs or the identification of appropriate genetic changes that may be useful for gene therapy. Other applications include soil ecology where it is desirable to be able to isolate DNA from any soil or tissue sample and to identify microorganisms present in the sample using probes from ribosomal DNA sequences from all known microorganisms. In fields such as science or pathology.
[0003]
Routine sequencing of DNA using electrophoresis is typically labor intensive and time consuming. Various alternatives to normal DNA sequencing have been proposed. One such alternative approach that utilizes an array of oligonucleotide probes synthesized by photolithography techniques is Pease et al., “Light-Generated Oligonucleotide Arrays for Rapid DNA Sequence Analysis”, Proc. Natl. Acad. Sci. USA, 91, 5022-5026, May 1994. In this approach, the surface of a solid support modified with a photolabile protecting group is illuminated through a photolithographic mask, producing reactive hydroxyl groups in the illuminated area. A 3′-activated deoxynucleoside protected at the 5′-hydroxyl position with a photolabile group is then applied to the surface so that coupling occurs at the site exposed to light. After capping and oxidation, the support is rinsed and the surface is illuminated through a second mask to expose additional hydroxyl groups for coupling. A second 5 ′ protected activated deoxynucleoside base is provided on the surface. Selective photodeprotection and coupling cycles are repeated to build a level of base until the desired set of probes is obtained. Using such photolithography techniques (the sequence of oligonucleotide probes at each site in the array is known) it may be possible to produce a dense miniaturized array of oligonucleotide probes. These probes are then studied for the complementary sequence of the target strand of DNA by detection of the target hybridized to a specific probe and the examination by appropriate fluorescence scanning microscopy, which is done by the use of a fluorescent marker coupled to the target. Can be used for A change in this method using polymer semiconductor photoresists (which are selectively patterned by photolithographic techniques rather than using photolabile 5 'protecting groups) is McGall et al., “Light-Directed Synthesis of High-Density Oligonucleotide Arrays Using Semiconductor Photoresists ", Proc. Natl. Acad. Sci. USA, 93, 13555-13560, November 1996, and GH McGall et al.,“ The Efficiency of Light-Directed Synthesis of DNA Arrays. on Glass Substrates ", Journal of the American Chemical Society 119, 22, 1997, 5081-5090.
The disadvantage of both of these approaches is that four different lithographic masks are required for each monomer base, thus the total number of different masks required is four times the length of the DNA probe sequence synthesized. That is. The high cost of producing the many precise photolithographic masks required and the multiple processing steps required to reposition the mask with each exposure contribute to relatively high costs and too long processing times.
[0004]
(Disclosure of the Invention)
According to the present invention, the synthesis of an array of DNA probe sequences, polypeptides, etc. is carried out quickly and effectively using a patterning method. The method can be automated and allow processing of one- or two-dimensional arrays of probes, including probe arrays that are computer controlled and custom-made for special studies. A lithographic mask is not required, thus eliminating significant costs and time delays associated with the manufacture of lithographic masks, avoiding time-consuming operations and multiple mask alignment during probe array processing methods.
In the present invention, a support having an active surface to which a DNA synthesis linker is applied is used to support a probe to be processed. In order to activate the active surface of the support to give a first level of base, a highly accurate two-dimensional light image is projected onto the support, illuminating these pixels in the array of support active surfaces, these Is activated and binds to the first base. Light incident on the pixels in the array to which the light is applied deprotects the OH groups and makes them available to bind to the base. After this development step, a liquid containing a suitable base is applied to the active surface of the support, and the selected base binds to the exposed site. The method is then repeated to attach another base to a different set of pixel positions until all of the elements of the two-dimensional array of support surfaces have the appropriate base attached to them. The base bound to the support is protected with one or more layers of photoresist that covers all of the bound base or chemicals that can bind to the base, and then a new array pattern is projected and the first new base Is imaged onto the support to activate the protective material in those pixels to which is added. These pixels are then exposed and a solution containing the selected base is applied to the array so that the bases bind at the exposed pixel locations. The method is then repeated for all other pixel positions at the second level of base. The described method may then be repeated for each desired level of bases until a complete selected two-dimensional array of probe sequences is completed.
[0005]
The image provides a suitable light source that provides light to a micromirror device comprising a two-dimensional array of electrically addressable micromirrors, each of which can be selectively tilted between one of at least two other positions. The image is formed on a support using an image forming apparatus. At one of the positions of each micromirror, the light from the light source incident on the micromirror is deflected away from the optical axis and away from the support, and the second of at least two positions of each micromirror. In this position, light is reflected along the optical axis and toward the support. The projection optics receives the light reflected from the micromirror and accurately images the micromirror on the active surface of the support. A collimating optical device may be used to collimate light from the light source into a beam that is directly applied to the micromirror array or beam splitter, which reflects a portion of the beam to the micromirror array and reflects it. The transmitted light is propagated from the micromirror array into the beam splitter. Light reflected directly from the micromirrors or propagated through the beam splitter is directed to the projection optical lens, which images the micromirror array onto the active surface of the support. The selectively addressable micromirrors in the micromirror array can either completely reflect or fully deflect the light applied to them, so that the image of the micromirror array is an “on” pixel and an “off” pixel. A very high contrast between. Micromirrors may also be indexed at more than two positions, in which case additional optical devices are provided to provide more than one support using a single micromirror array device. May allow exposure. In addition, micromirrors can reflect light of any wavelength without causing damage to them, allowing short wavelength light, including light in the ultraviolet to near ultraviolet range, to be utilized from the light source.
[0006]
The micromirror array is operated under computer control to provide the appropriate pixel address signal to the micromirror array so that the appropriate micromirrors are in their “reflective” or “deflection” position. A micromirror array pattern suitable for each activation step at each level of base added to the probe is programmed into the computer controller. Thus, the computer controller controls the sequencing of the image provided by the micromirror array in cooperation with the reagent provided on the support.
In one embodiment, the support may be transparent, allowing the image of the micromirror array to be projected onto the surface of the support opposite the active surface. The support is an enclosure that seals the active surface of the array and may be mounted within the flow cell, and appropriate reagents are flowed through the flow cell in the appropriate order onto the active surface of the array to build probes in the array. Make it possible.
Further objects, features and advantages of the present invention will become apparent from the following detailed description when taken in conjunction with the accompanying drawings.
[0007]
(Best Mode for Carrying Out the Invention)
Referring to the drawings, an exemplary apparatus that can be used for DNA probe array synthesis, polypeptide synthesis, etc. is shown generally at 10 in FIG. And the support 12 to be made. For the configuration shown in FIG. 1, the support has an exposed entrance surface 14 and an opposite active surface 15 on which a two-dimensional array of nucleotide sequence probes 16 is processed. For illustrative purposes, the support 12 is shown in the figure with a flow cell enclosure 18 attached to the support 12 that seals the volume 19 through which reagents are provided through the inlet 20 and outlet 21. However, the support 12 faces the imaging device 11 and the active surface 15 of the support sealed in a reaction chamber flow cell with a transparent window that allows light to be projected onto the active surface. May be used. The present invention may also use an opaque or porous support. Reagents may be provided to mouths 20 and 21 from a conventional base synthesizer (not shown in FIG. 1).
[0008]
The image forming apparatus 11 receives a light source 25 (for example, an ultraviolet source or a near ultraviolet source, for example, a mercury arc lamp), an output beam 27 from the light source 25, and selectively passes only a desired wavelength (for example, a 365 nm Hg line). It includes an optional filter 26 and a condenser lens 28 for forming a collimated beam 30. Filters or other devices for making source light monochromatic, such as diffraction gratings, dichroic mirrors, and prisms, may also be used in addition to transmission filters and are generally referred to herein as “filters”. The beam 30 is projected onto a beam splitter 32, which reflects a portion of the beam 30 onto the beam 33, which is projected onto the two-dimensional micromirror array device 35. The micromirror array device 35 has a two-dimensional array of individual micromirrors 36 that are tilted in one of at least two directions in response to control signals supplied to the array device 35, respectively. The control signal is given from the computer controller 38 of the control line 39 to the micromirror array device 35. The micromirror 36 is such that at the first position of the mirror, a portion of the incident beam of light 33 impinging on the individual micromirror 36 is deflected in an oblique direction of the incident beam 33 as indicated by the arrow 40. able to make. At the second position of the mirror 36, light from the beam 33 impinging on such a mirror at such second position is retro-reflected parallel to the beam 33 as indicated by the arrow 41. The light reflected from each of the mirrors 36 constitutes an individual beam 41. Multiple beams 41 enter beam splitter 32, pass through the beam splitter with reduced intensity, and then enter projection optics 44, including, for example, lenses 45 and 46 and adjustable iris 47. The projection optics 44 can be used to image the pattern of the micromirror array 35 as represented by the individual beams 41 (and the dark areas between these beams) on the active surface 15 of the support 12. The outgoing beam 41 is guided along the main optical axis of the image forming apparatus 11 extending between the micromirror device and the support. The support 12 in the form shown in FIG. 1 is transparent, for example formed from fumed silica or soda lime glass or quartz, and is thus projected onto it, exemplified by the line labeled 49. The transmitted light passes through the support 12 with substantially no attenuation or diffusion.
[0009]
A preferred micromirror array 35 is a digital micromirror device (DMD) commercially available from Texas Instruments. These devices are micromirrors (each of which is essentially a square having sides of 10-20 μm in length, which can form a patterned beam of light by electrically addressing the micromirrors in the array. ). Such DMD devices are typically used for video projection and come in various array sizes such as 640x800 micromirror elements (512,000 pixels), 640x480 (VGA; 307,200 pixels), 800x600 (SVGA; 480,000 pixels), and 1024x768. (786,432 pixels). Such arrays are described in the following literature and patents: Larry J. Hornbeck, “Digital Light Processing and MEMs: Reflecting the Digital Display Needs of the Networked Society”, SPIE / EOS European Symposium on Lasers, Optics, and Vision for Productivity and Manufacturing I, Besancon, France, June 10-14, 1996, and U.S. Pat. Nos. 5,096,279, 5,535,047, 5,583,688, and 5,600,383. The micromirror 36 of such a device can reflect light of commonly usable wavelengths, including ultraviolet and near ultraviolet, in an effective manner without causing damage to the mirror itself. The window of the enclosure for the micromirror array preferably has an antireflection coating optimized thereon for the wavelength of light used. Utilizing a 600x800 array of commercially available micromirrors that encode 480,000 pixels, with a typical micromirror device size of 16 microns per mirror surface and a pitch in an array of 17 microns, a total of 13,600 microns x 10,200 microns Gives micromirror array dimensions. By using a reduction factor of 5 in the optical system 44, which is a typical and easily achievable value for a lithographic lens, the size of the image projected on the support 12 thus has a resolution of about 2 microns, Approximately 2,220 microns x 2040 microns. Larger images can be exposed to the support 12 by utilizing multiple parallel exposure (by stepping the flow cell 18 or image projector 11) or by using a larger micromirror array. If desired, it is possible not only to reduce the image of the support but also to form a one-to-one image without enlarging.
[0010]
The projection optical device 44 may be of a normal design. This is because the image formed is relatively large and well away from the diffraction limit. Lenses 45 and 46 concentrate the light in beam 41 passed through adjustable iris 47 onto the active surface of the support. Projection optics 44 and beam splitter 32 are from a primary optical axis (the central axis of projection optics 44 in which beam 41 is parallel), indicated by a beam labeled 40 (eg, 10 ° off axis). Positioned so that light deflected by the micromirror array away enters the entrance optics of the projection optics 44 (typically 0.5 / 5 = 0.1; 10 ° is 0.17 aperture substantially larger than 0.1) Equivalent to The iris 47 is used to adjust the effective aperture and to ensure that unwanted light (particularly the beam 40 off axis) is not propagated to the support. A resolution as small as 0.5 microns can be obtained with such an optical system. For manufacturing applications, the micromirror array 35 is preferably placed in the object focal plane of a lithographic I-line lens optimized for 365 nm. Such lenses typically operate with a numerical aperture (NA) of 0.4 to 0.5 and have a large field capacity.
[0011]
The micromirror array device 35 may be formed of a single line of micromirrors that are stepped in the scanning system (eg, having 2,000 mirror elements in one line). In this manner, the height of the image is fixed by the line length of the micromirror array, but the width of the image that can be projected on the support 12 is not substantially limited. By moving the stage 18 with the support 12, the mirror can be cycled at each index position of the support to form an image pattern in each new line that is imaged on the support active surface.
Various approaches may be utilized for processing the DNA probe 16 on the support 12, employing microlithographic techniques. In the “direct light processing approach”, the glass support 12 is coated with a layer of a drug capable of binding nucleotide bases. Light is applied by projection system 11 to deprotect the OH groups of the support and make them available to attach to the base. After development, the appropriate nucleotide base is flowed over the active surface of the support and attached to the selected site using conventional phosphoramidite DNA synthesis chemistry. The method is then repeated to attach another base to a different set of positions. The method is simple and the number of permutations increases exponentially when a combinatorial approach is used. The resolution limit is represented by the linear response of the deprotection mechanism. Because of the resolution limitations obtained with this method, methods based on photoresist technology may be used instead, as described, for example, in the above-referenced article by McGall et al. In the indirect photoprocessing approach, compatible chemistry exists in a two-layer resist system, where, for example, the first layer of polyimide acts as an underlying chemical protection, while the top imaging resist is based on epoxy. It is a system. The imaging process is common to both methods, the main requirement is that the wavelength of light used in the imaging method does not excite transitions (chemical changes) in nucleotide bases (these are particularly sensitive at 280 nm). That is long enough. Therefore, wavelengths longer than 300 nm should be used. 365nm is the mercury I-line, which is most commonly used for wafer lithography.
[0012]
Another form of array synthesizer 10 is shown in the schematic diagram of FIG. In this arrangement, the beam splitter 32 is not used and the light source 25, optional filter 26, and condenser lens 28 are mounted at an angle to the main optical axis (eg, 20 ° to the axis) to direct the beam of light 30. Projected on an array of micromirrors 36 at an angle. Micromirror 36 is oriented to reflect light 30 to off-axis beam 40 at the first position of the mirror and to beam 41 along the major axis at the second position of each mirror. In other respects, the array synthesis apparatus of FIG. 2 is the same as the apparatus of FIG.
[0013]
A more detailed view of a preferred array synthesizer using the off-axis projection arrangement of FIG. 2 is shown in FIG. In the apparatus of FIG. 3, a light source 25 (eg, a 1000 W Hg arc lamp, Oriel 6287, 66021), powered by a power source 50 (eg, Oriel 68820), is used as the light source containing the desired ultraviolet wavelength. The Filter system 26 includes, for example, a dichroic mirror (eg, Oriel 66226) that is used to absorb infrared light and selectively reflect light in the wavelength range of 280 nm to 400 nm. A water cooled liquid filter (eg, Oriel 6127) with deionized water is used to absorb the remaining infrared radiation. A colored glass filter (Oriel 59810) or interference filter (Oriel 56531) is used to select the 365 nm line of the Hg lamp 25 having a 50% bandwidth of 50 nm or 10 nm, respectively. An F / l two-element fused silica condenser (Oriel 66024) is used as condenser 28 to form a caller illumination system with two flat eyepiece lenses 52 (Meles Griot 01LQP033 and Meles Griot 01LQP023). This illumination system produces an approximately collimated uniform beam 30 of 365 nm light with a diameter just large enough to contain the 16 mm × 12 mm active region of the micromirror array device 35. This beam 30 is incident on the device 35 at an angle of 20 ° as measured from perpendicular to the surface of the device. The micromirror array device 35 is arranged about 700 mm away from the last filter. When the micromirror is in the first position, the light in beam 30 is deflected downward from the system. For example, in this micromirror device, the mirrors may be at an angle of −10 ° to the surface of the micromirror at their first position and reflect light well away from the optical axis. When the micromirror is adjusted to be deflected at a second position, for example at an angle of + 10 ° with respect to the plane of the micromirror, it is reflected from such a micromirror at the second position. Light appears in beam 41 perpendicular to the plane of the micromirror array. The pattern formed by the light reflected from the micromirrors in their second position is then sealed in the flow cell 18 using a telecentric imaging system that includes two double lenses 45 and 46 and an adjustable aperture 47. An image is formed on the active surface 15 of the finished glass support 12. Each of the double lenses 45 and 46 includes a pair of flat lenses (eg, Meles Griot 01LQP033 and 01LQP037) that are brought together so that the curved surfaces are substantially touched. The first double lens is oriented so that the short focal length (01LQP033) side faces the micromirror array device 35, and the second double lens is oriented so that the long focal length (01LQP037) side is the micromirror array device 35. Oriented to face. Double lenses including the same lens may be used, in which case either side may face the micromirror array device. An adjustable aperture 47, also referred to as a telecentric aperture, is placed on the back focal plane of the first double lens. It is used to change the angle acceptance of the optical system. Small aperture diameters correspond to improving contrast and resolution, but image intensity was reduced accordingly. As shown in FIG. 3, a normal DNA synthesizer 55 supplied with the necessary chemicals is connected to the flow cell 18 by tubes 20 and 21 under independent control or under the control of a computer 38. Of the desired sequence. A typical diameter for the aperture 47 is about 30 nm. A ray diagram showing the path of light in lenses 45 and 46 is shown in FIG. 4 for this type of refractive optical system. Shown is a fan of rays that occur at the center of the object (micromirror device surface), at the edges, and at intermediate positions. The optical system forms an inverted image of the surface of the micromirror array device.
[0014]
Another embodiment of an array synthesizer that uses reflective optics is shown in FIG. An exemplary system is a 1000 W Hg arc lamp as a light source with a filter system formed from a dichroic mirror (eg, Oriel 66228) that absorbs infrared light and selectively reflects light in the wavelength range of 350 nm to 450 nm. 25 (for example, Oriel 6287, 66021) is used. An F / l two-element fused silica condenser lens (Oriel 66024) is used to produce an approximately collimated beam of light 30 that includes 365 nm lines but excludes undesirable wavelengths below 300 nm. A caller illumination system may also be used in the apparatus of FIG. 5 if necessary to increase uniformity and strength. Beam 30 has a micromirror active area of about 16 mm x 12 mm and is incident on a micromirror array device 35 located about 210 nm from the nozzle of UV source 25, and beam 30 is perpendicular to the plane of the array. It collides with the plane of the micromirror device 35 at an angle of 20 °. At the first position of the micromirror, for example, light reflected from the micromirror at -10 ° to the plane of the array is guided from the system, while the second position, for example + Light from the 10 ° micromirror is directed in the beam 41 towards a reflective telecentric imaging system that includes a concave mirror 60 and a convex mirror 61. Both mirrors are spherical and preferably have an enhanced UV coating for high reflectivity. After reflection from the mirrors 60 and 61, the beam 41 may enter another plane mirror 63, which deflects the beam toward the flow cell 18. The light reflected from the micromirrors is imaged on the active surface of the glass support sealed in the flow cell 18. A telecentric aperture (not shown in FIG. 5) may be placed in front of the convex mirror. Beam 41 first hits the concave mirror, then hits the convex mirror, then hits the concave mirror again, and plane mirror 63 deflects the light by 90 ° if necessary to guide it to flow cell 18 May be used. For the system shown, the concave mirror 60 may have a diameter of 152.4 mm and a spherical mirror surface radius of 304.8 mm (ES F43561), and the convex mirror may have a diameter of 25 mm and 152.94 mm (ES F45625). ) Spherical mirror surface radius. Ideally, the radius of curvature of the concave mirror is twice that of the convex mirror. Such reflective optics are well known and are commonly used in optical lithography in “micro-aligned” type systems. For example, A. Offner, “New Concepts in Projection Mask Aligners”, Optical Engineering, 14, 130-132 (1975), and RT Kerth et al., “Excimer Laser Projection Lithography on a Full-Field Scanning Projection Systems”, IEEE See Electron Device Letters, EDL-7 (5), 299-301 (1986).
[0015]
FIG. 6 shows image formation for the optical system of FIG. FIG. 6 shows a fan of light rays generated at the center, end, and middle positions of the object (micromirror array device). The light beam is first reflected from the concave mirror 60, then from the convex mirror 61, and then again from the concave mirror 60 to form an inverted image of the surface of the micromirror array device. The plane mirror 63 is not included in the diagram of FIG. A telecentric aperture (not shown) may be placed in front of the convex mirror.
Both refractive and reflective optical systems are preferably “symmetric” and erasure minimizes aberrations such as coma and spherical aberration. The above reflection system has a high aperture number that produces high strength. Both the telecentric optics of FIGS. 3 and 5 are 1: 1 imaging systems. The reflection system not only eliminates chromatic aberration and provides high resolution, but also has the potential advantage of being compact and inexpensive. A preferred system for performing 1: 1 imaging would be a Wine-Dyson type system with a concave mirror and a lens and prism. It has a very high caliber that enhances strength. For example, FN Goodall et al., “Excimer Laser Photolitography with 1: 1 Wynne-Dyson Optics”, SPIE 922, Optical / Laser Microlithography, 1988, and B. Ruff et al. “Broadband Deep-UV High NA Photolithography System”, SPIE 1088. See Volume, Optical / Laser Microlithography II (1989).
[0016]
A more detailed view of a reaction chamber flow cell 18 that can be used with the apparatus of the present invention is shown in FIGS. The exemplary flow cell 18 in these figures includes an aluminum housing 70 held together by bolts 71 having an inlet 73 connected to the inlet line 20 and an outlet 75 connected to the outlet line 21. As shown in the cross-sectional view of FIG. 8, the housing 70 includes a lower base 78 and an upper cover portion 79 that are secured together on a support by bolts 71. The support 12, for example, a transparent glass slide, has an upper plate 79 and a cylindrical gasket 81 (for example, Cal Let's TM Which is in turn formed between the non-reactive base block 82 (eg Teflon) TM The inlet channel 85 extends from the inlet 73 to a sealed reaction chamber 88 formed between the support 12 and the base block 82 (which is sealed by a gasket), and an outlet channel 89 Extending from reaction chamber 88 to outlet 75. Bolt 71 may or may not be screwed, support 12 is removably secured between the cover part and the base, and the support is replaced with minimal displacement of the base of the flow cell. Allows to be done. Preferably, as shown in FIG. 8, a rubber gasket 90 is attached to the bottom of the plate 79 to engage the support in the peripheral region and apply pressure to the support against the gasket 81. If desired, the flow cell may also be used as a hybridization chamber during reading.
[0017]
An exemplary method of forming a DNA probe is shown with respect to the schematic of FIGS. 9-14. FIG. 9 shows the coating of the support 12 with the silane layer 95 forming the active surface 15 by the photolabile linker molecule MENPOC-HEG coated on the silane layer using conventional phosphoramidite chemistry. MENPOC-HEG-CEP is 18-O-[(R, S)-(1- (3,4- (methylenedioxy) -6-nitrophenyl) ethoxy) carbonyl] -3,6,9,12,15 , 18-Hexaoxaoctadec-1-yl O'-2-cyanoethyl-N, N-diisopropyl phosphoramidite. The silane layer was made from N- (3- (triethoxysilyl) -propyl) -4-hydroxybutyramide. In the process shown in FIG. 9, the support will be exposed to light and the active free OH groups will be exposed in the areas exposed to light.
FIG. 10 shows photodeprotection of the MENPOC-HEG linker and generation of free OH groups in the region 100 exposed to light. Figure 11 shows the fluorprime to the free OH group generated from the photodeprotection of MENPOC-HEG TM Fluorescein amidite coupling is shown. FIG. 12 shows the coupling of DMT-nucleotide to the free OH group generated from the photodeprotection of MENPOC-HEG linker. FIG. 13 shows the process of acid deprotection of DMT-nucleotide in the region 100 exposed to light. FIG. 14 shows the hybridization of a fluorescein labeled poly-A probe with a poly-T oligonucleotide synthesized from DMT-nucleotide-CEP.
While the invention is not limited to the specific embodiments shown herein by way of example, it will be understood to encompass all such modifications as fall within the scope of the claims.
[Brief description of the drawings]
FIG. 1 is a schematic diagram of an array synthesizer of the present invention.
FIG. 2 is a schematic diagram of another array synthesizer of the present invention.
FIG. 3 is a more detailed schematic diagram of a general telecentric array synthesizer of the present invention.
4 is an exemplary ray diagram for the refractive optical device of the apparatus of FIG. 3;
FIG. 5 is a schematic diagram of yet another embodiment of the array synthesizer of the present invention in which a telecentric reflective optical device is utilized.
6 is an exemplary ray diagram for the reflective optical device of the apparatus of FIG. 5;
FIG. 7 is a plan view of a reaction chamber flow cell that can be used in the array synthesizer of the present invention.
8 is a cross-sectional view of the reaction chamber flow cell of FIG. 7 taken generally along line 8-8 of FIG.
FIG. 9 is a schematic showing the coating of a support with a photolabile linker molecule.
FIG. 10 is a schematic showing photodeprotection of linker molecules and generation of free OH groups.
FIG. 11 is a schematic showing the coupling of a marker to a free OH group generated by photodeprotection of a linker molecule.
FIG. 12 is a schematic showing the coupling of DMT-nucleotides to free OH groups generated from photodeprotection of linker molecules.
FIG. 13 is a schematic showing acid deprotection of DMT-nucleotides.
FIG. 14 is a schematic showing the hybridization of a fluorescein labeled poly-A probe with a poly-T oligonucleotide synthesized from DMT-nucleotide-CEP.

Claims (15)

(a)アレイを形成し得る活性表面を有する支持体、
(b)該支持体の活性表面を密閉し、かつ該支持体の活性表面に流される試薬をフローセルに適用するための口部を有するフローセル、
(c)(1)光ビームを与える光源、
(2)該光ビームを前記光源から受け取り、かつ電気的にアドレス可能であるとともに、夫々が少なくとも二つの別の位置の一つの間で選択的に傾斜し得るミクロミラーのアレイから形成されたミクロミラー装置であって、夫々のミクロミラーの位置の一つにおいて、前記ミクロミラーに入射する前記光源からの光が光学軸から離れて偏向され、かつ該ミクロミラーの少なくとも二つの位置の第二の位置で光が光学軸に沿って反射されるミクロミラー装置、及び
(3)光学軸に沿って該ミクロミラーから反射された光を受け取り、かつ該ミクロミラーのパターンを支持体の活性表面に像形成する映写光学装置であって、２つの二重集束レンズと調節可能なアイリスとを含み、第一の二重レンズが光を第一の二重レンズの逆焦点面に位置した調節可能なアイリス中に通し、第二の二重レンズが該アイリス中を通された光を受け取り、かつその光を支持体の活性表面に集中させる映写光学装置、
を含む、支持体活性表面に映写された高い精度の二次元の光像を与える像形成装置、
を含むことを特徴とする装置。(a) a support having an active surface capable of forming an array;
(b) a flow cell having a mouth for sealing the active surface of the support and applying a reagent to be flowed to the active surface of the support to the flow cell;
(c) (1) a light source that provides a light beam,
(2) a micro that is formed from an array of micromirrors that receive the light beam from the light source and are electrically addressable and each can be selectively tilted between one of at least two other positions. A mirror device, wherein at one of the positions of each micromirror, the light from the light source incident on the micromirror is deflected away from the optical axis and a second of at least two positions of the micromirror A micromirror device in which light is reflected along the optical axis at a position; and
(3) A projection optical device that receives light reflected from the micromirror along the optical axis and forms an image of the micromirror pattern on the active surface of the support, adjusted with two double focusing lenses A first double lens that passes light through an adjustable iris located in the back focal plane of the first double lens, and a second double lens that is passed through the iris. Projection optical device that receives the reflected light and concentrates the light on the active surface of the support,
An image forming apparatus for providing a high-precision two-dimensional optical image projected on a support active surface,
The apparatus characterized by including. 前記ミクロミラー装置がミクロミラーの二次元アレイから形成される請求の範囲第１項記載の装置。The apparatus of claim 1 wherein said micromirror device is formed from a two-dimensional array of micromirrors. 前記光源からのビームを平行にして該ミクロミラーアレイから該支持体に延びる主光学軸に斜めの角度で該ミクロミラーアレイに映写された平行にされたビームを得るためのレンズを含み、かつ夫々のミクロミラーの一つの位置で、光が光学軸に沿って該映写光学装置を通って支持体に反射され、又、夫々のミクロミラーの第二の位置で、該光源からの光が映写系の主軸から離れた角度で、かつ該支持体から離れて反射される請求の範囲第１項記載の装置。A lens for collimating the beam from the light source to obtain a collimated beam projected onto the micromirror array at an oblique angle to a main optical axis extending from the micromirror array to the support, and At one position of the micromirror, light is reflected along the optical axis through the projection optics to the support, and at the second position of each micromirror, the light from the light source is projected into the projection system. 2. The apparatus of claim 1 wherein the apparatus is reflected at an angle away from the main axis of the substrate and away from the support. 前記光源が出力ビームを平行にするレンズに出力ビームを与え、かつミクロミラーアレイと映写光学装置の間に配置されたビームスプリッターを含み、かつ平行にされたビームを光源から受け取り、そのビームスプリッターはビームの一部を該ミクロミラーアレイに反射し、かつ該ミクロミラーアレイから前記映写光学装置を通って該支持体に延びる装置の主光学軸に沿って該ミクロミラーアレイから反射された光を受け取り、ビームスプリッターがミクロミラーからの光をその中を通って映写光学装置に部分的に通して支持体の活性表面で像形成される請求の範囲第１項記載の装置。The light source provides an output beam to a lens that collimates the output beam, and includes a beam splitter disposed between the micromirror array and the projection optics, and receives the collimated beam from the light source, the beam splitter comprising: Receiving a portion of the beam reflected from the micromirror array along the main optical axis of the device that reflects from the micromirror array and extends from the micromirror array through the projection optics to the support The apparatus of claim 1 wherein the beam splitter is imaged at the active surface of the support through the light from the micromirrors partially through the projection optics. 前記光源からの光を受け取り、かつ所望の波長のみを該ミクロミラーアレイに選択的に通すフィルターを更に含む請求の範囲第１項記載の装置。The apparatus of claim 1 , further comprising a filter that receives light from the light source and selectively passes only a desired wavelength through the micromirror array. 前記支持体が透明であり、かつ像形成装置からの光が透明支持体中を通されて光を像形成装置から初期に受け取る表面とは反対である支持体の活性表面で像形成される請求の範囲第１項記載の装置。Claims wherein the support is transparent and the light from the imaging device is imaged on the active surface of the support opposite to the surface that is passed through the transparent support and initially receives light from the imaging device. A device according to claim 1, wherein 前記ミクロミラー装置に連結されたコンピュータを更に含んでコマンドシグナルを与えて該ミクロミラーアレイ中のミラーの偏向を調節して映写のための所望のパターンを支持体に与える請求の範囲第１項記載の装置。Desired pattern applied to the support range first claim of claim for projection by adjusting the deflection of the mirror in the micro mirror array giving further comprise command signals the computer that is connected to the micro-mirror device Equipment. 前記光源により与えられた光が紫外波長から近紫外波長までの範囲である請求の範囲第１項記載の装置。The apparatus according to claim 1, wherein the light provided by the light source is in a range from an ultraviolet wavelength to a near ultraviolet wavelength. 紫外及び近紫外の波長を選択的に通し、かつ赤外を含む更に長い波長を遮断する、光源からの光を受け取るフィルターを含む請求の範囲第８項記載の装置。9. The apparatus of claim 8 including a filter for receiving light from a light source that selectively passes ultraviolet and near ultraviolet wavelengths and blocks longer wavelengths including infrared. 前記フィルターが選ばれた波長を反射し、かつ遮断される波長を通すダイクロイックミラーを含む請求の範囲第９項記載の装置。The apparatus of claim 9 wherein the filter includes a dichroic mirror that reflects selected wavelengths and passes wavelengths that are blocked. 前記支持体の活性表面で像形成される前記ミクロミラーのパターンが該ミクロミラーのアレイのサイズに対してサイズを減少される請求の範囲第１項記載の装置。The apparatus of claim 1 wherein the pattern of micromirrors imaged on the active surface of the support is reduced in size relative to the size of the array of micromirrors. 前記光源とコーラー照明系を形成する前記ミクロミラー装置との間に屈折レンズを含む請求の範囲第１項記載の装置。The apparatus according to claim 1 , further comprising a refractive lens between the light source and the micromirror device forming a caller illumination system. 試薬をフローセルに供給するために連結されたDNA合成装置を含む請求の範囲第１項記載の装置。The apparatus of claim 1 including a DNA synthesizer coupled to supply reagents to the flow cell. 前記フローセルが下部ベースを含むハウジングを有し、かつ上部カバー部分がベースに取り付けられたガスケットであり、前記支持体が透明ガラススライドであり、該スライドが、前記上部カバー部分と該ベースとの間に固定されて、該支持体とガスケットとによりシールされるベースの間にシールされた反応チャンバーを形成し、かつチャンネルが該ハウジング中を入口から前記反応チャンバーに、又、該反応チャンバーから出口に延び、該支持体の活性表面が、前記シールされた反応チャンバーに面する請求の範囲第１項記載の装置。The flow cell has a housing including a lower base, and the upper cover portion is a gasket attached to the base, the support is a transparent glass slide, and the slide is between the upper cover portion and the base. Fixed to the base and sealed between the support and the gasket to form a sealed reaction chamber, and a channel through the housing from the inlet to the reaction chamber and from the reaction chamber to the outlet. The apparatus of claim 1 wherein the active surface of the support extends and faces the sealed reaction chamber. 前記支持体を該下部ベースと該上部カバー部分との間に取り外し可能に固定して、該支持体が交換されることを可能にするための装置を含む請求の範囲第４項記載の装置。The apparatus of claim 4 including an apparatus for removably securing the support between the lower base and the upper cover portion to allow the support to be replaced.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023200259A1 (en) * 2022-04-13 2023-10-19 고려대학교 산학협력단 System and method for precise optical synthesis of dna

Families Citing this family (173)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5143854A (en) * 1989-06-07 1992-09-01 Affymax Technologies N.V. Large scale photolithographic solid phase synthesis of polypeptides and receptor binding screening thereof
US5846717A (en) * 1996-01-24 1998-12-08 Third Wave Technologies, Inc. Detection of nucleic acid sequences by invader-directed cleavage
US7527928B2 (en) * 1996-11-29 2009-05-05 Third Wave Technologies, Inc. Reactions on a solid surface
US7432048B2 (en) * 1996-11-29 2008-10-07 Third Wave Technologies, Inc. Reactions on a solid surface
US20040035690A1 (en) * 1998-02-11 2004-02-26 The Regents Of The University Of Michigan Method and apparatus for chemical and biochemical reactions using photo-generated reagents
US6426184B1 (en) * 1998-02-11 2002-07-30 The Regents Of The University Of Michigan Method and apparatus for chemical and biochemical reactions using photo-generated reagents
EP2259046A3 (en) * 1998-02-23 2011-11-30 Wisconsin Alumni Research Foundation Method for synthesis of arrays of dna probes
US6271957B1 (en) * 1998-05-29 2001-08-07 Affymetrix, Inc. Methods involving direct write optical lithography
WO1999063385A1 (en) * 1998-06-04 1999-12-09 Board Of Regents, The University Of Texas System Digital optical chemistry micromirror imager
DE19940751A1 (en) * 1998-08-28 2000-03-02 Febit Ferrarius Biotech Gmbh Apparatus for detecting light emissions comprises light-emitting matrix facing light-detection matrix, which together sandwich test substance
US6801353B2 (en) * 1999-02-01 2004-10-05 Production Resource Group Inc. Pixel based gobo record control format
EP1180111A4 (en) * 1999-02-10 2003-03-26 Macrogen Inc Method and apparatus for compound library preparation using optical modulator
DE19932487A1 (en) * 1999-07-09 2001-02-08 Epigenomics Gmbh Photolithography illumination of biological matter uses an elastic mirror surface with controlled distortion to give structured diffracted light for illuminated and unlit zones at the target surface
US6472588B1 (en) * 1999-09-10 2002-10-29 Texas Tech University Transgenic cotton plants with altered fiber characteristics transformed with a sucrose phosphate synthase nucleic acid
US6315958B1 (en) 1999-11-10 2001-11-13 Wisconsin Alumni Research Foundation Flow cell for synthesis of arrays of DNA probes and the like
DE10027119A1 (en) * 2000-05-23 2001-12-06 Epigenomics Ag Photolithographic exposure unit for e.g. manufacturing DNA chips, scans target areas of biological material selectively with highly-focused flying spot illumination
DE10027524A1 (en) * 2000-06-02 2001-12-13 Max Planck Gesellschaft Device and method for processing substrate-bound samples
WO2002002227A2 (en) * 2000-07-03 2002-01-10 Xeotron Corporation Devices and methods for carrying out chemical reactions using photogenerated reagents
WO2002004597A2 (en) * 2000-07-07 2002-01-17 Nimblegen Systems, Inc. Method and apparatus for synthesis of arrays of dna probes
US6567163B1 (en) 2000-08-17 2003-05-20 Able Signal Company Llc Microarray detector and synthesizer
US6545758B1 (en) 2000-08-17 2003-04-08 Perry Sandstrom Microarray detector and synthesizer
DE10051396A1 (en) 2000-10-17 2002-04-18 Febit Ferrarius Biotech Gmbh An integrated synthesis and identification of an analyte, comprises particles immobilized at a carrier to be coupled to receptors in a structured pattern to give receptor arrays for biochemical reactions
US6706867B1 (en) 2000-12-19 2004-03-16 The United States Of America As Represented By The Department Of Health And Human Services DNA array sequence selection
US20020075490A1 (en) * 2000-12-20 2002-06-20 Affymetrix, Inc. System and method for addressable light-directed microarray printing
US7211654B2 (en) 2001-03-14 2007-05-01 Regents Of The University Of Michigan Linkers and co-coupling agents for optimization of oligonucleotide synthesis and purification on solid supports
US20020160427A1 (en) * 2001-04-27 2002-10-31 Febit Ag Methods and apparatuses for electronic determination of analytes
CA2447240C (en) 2001-05-18 2013-02-19 Wisconsin Alumni Research Foundation Method for the synthesis of dna sequences
US7049049B2 (en) * 2001-06-27 2006-05-23 University Of South Florida Maskless photolithography for using photoreactive agents
WO2003008943A1 (en) * 2001-07-19 2003-01-30 Tufts University Optical array device and methods of use thereof for screening, analysis and manipulation of particles
US20030087298A1 (en) * 2001-11-02 2003-05-08 Roland Green Detection of hybridization on oligonucleotide microarray through covalently labeling microarray probe
US6790620B2 (en) * 2001-12-24 2004-09-14 Agilent Technologies, Inc. Small volume chambers
US7037659B2 (en) * 2002-01-31 2006-05-02 Nimblegen Systems Inc. Apparatus for constructing DNA probes having a prismatic and kaleidoscopic light homogenizer
US7422851B2 (en) * 2002-01-31 2008-09-09 Nimblegen Systems, Inc. Correction for illumination non-uniformity during the synthesis of arrays of oligomers
US20040126757A1 (en) * 2002-01-31 2004-07-01 Francesco Cerrina Method and apparatus for synthesis of arrays of DNA probes
US7157229B2 (en) 2002-01-31 2007-01-02 Nimblegen Systems, Inc. Prepatterned substrate for optical synthesis of DNA probes
US7083975B2 (en) * 2002-02-01 2006-08-01 Roland Green Microarray synthesis instrument and method
US20030186301A1 (en) * 2002-03-25 2003-10-02 The Regents Of The University Of California Constructing user-defined, long DNA sequences
US7452666B2 (en) 2002-03-25 2008-11-18 Lawrence Livermore National Security, Llc Synthesis of DNA
US20030180781A1 (en) * 2002-03-25 2003-09-25 The Regents Of The University Of California Constructing very long DNA sequences from synthetic DNA molecules
US20030228598A1 (en) * 2002-03-25 2003-12-11 The Regents Of The University Of California DNA/RNA as a write/read medium
US20070275411A1 (en) 2006-05-25 2007-11-29 Mcgall Glenn H Silane mixtures
US7332273B2 (en) * 2002-06-20 2008-02-19 Affymetrix, Inc. Antireflective coatings for high-resolution photolithographic synthesis of DNA arrays
EP1552003A4 (en) * 2002-06-28 2007-06-13 Rosetta Inpharmatics Llc Methods to assess quality of microarrays
DE10230320A1 (en) * 2002-07-05 2004-02-05 Marcel Rogalla Programmable illumination device for high resolution, massively parallel, spatial Systhese and analysis of microarrays
EP1573366B1 (en) * 2002-08-24 2016-11-09 Chime Ball Technology Co., Ltd. Continuous direct-write optical lithography
US7563600B2 (en) 2002-09-12 2009-07-21 Combimatrix Corporation Microarray synthesis and assembly of gene-length polynucleotides
AU2003270898A1 (en) * 2002-09-27 2004-04-19 Nimblegen Systems, Inc. Microarray with hydrophobic barriers
US20040110212A1 (en) * 2002-09-30 2004-06-10 Mccormick Mark Microarrays with visual alignment marks
US7482170B2 (en) * 2002-09-30 2009-01-27 Roche Nimblegen, Inc. Parallel loading of arrays
JP2006500954A (en) * 2002-10-01 2006-01-12 ニンブルゲン システムズ インコーポレイテッド A microarray with multiple oligonucleotides in a single array feature
US8069676B2 (en) 2002-11-13 2011-12-06 Deka Products Limited Partnership Water vapor distillation apparatus, method and system
US7090979B2 (en) * 2002-11-22 2006-08-15 The Regents Of The University Of California Derivatized versions of ligase enzymes for constructing DNA sequences
US7871799B2 (en) * 2002-11-22 2011-01-18 Lawrence Livermore National Security, Llc Sequential addition of short DNA oligos in DNA-polymerase-based synthesis reactions
SG121829A1 (en) * 2002-11-29 2006-05-26 Asml Netherlands Bv Lithographic apparatus and device manufacturing method
US20040115336A1 (en) * 2002-12-12 2004-06-17 Industrial Technology Research Institute Method of fabricating a grating-based optical biosensor
US20040115654A1 (en) * 2002-12-16 2004-06-17 Intel Corporation Laser exposure of photosensitive masks for DNA microarray fabrication
US6819469B1 (en) * 2003-05-05 2004-11-16 Igor M. Koba High-resolution spatial light modulator for 3-dimensional holographic display
US7061591B2 (en) * 2003-05-30 2006-06-13 Asml Holding N.V. Maskless lithography systems and methods utilizing spatial light modulator arrays
US6989920B2 (en) * 2003-05-29 2006-01-24 Asml Holding N.V. System and method for dose control in a lithographic system
EP1706488B1 (en) * 2004-01-12 2013-07-24 Roche NimbleGen, Inc. Method of performing pcr amplification on a microarray
ES2292110T3 (en) * 2004-01-26 2008-03-01 Nimblegen Systems, Inc. COMPUTER PROGRAM TO HELP IN THE IDENTIFICATION OF SNPS (POLYMORPHISMS OF A SINGLE NUCLEOTIDE) WITH MICROFORMATIONS.
JP4095968B2 (en) * 2004-02-06 2008-06-04 株式会社日立ハイテクノロジーズ Liquid dispensing device, automatic analyzer using the same, and liquid level detecting device
CA2557841A1 (en) * 2004-02-27 2005-09-09 President And Fellows Of Harvard College Polony fluorescent in situ sequencing beads
CA2562285A1 (en) * 2004-04-08 2006-03-16 Nimblegen Systems, Inc. Comparative genomic resequencing
US7072500B2 (en) * 2004-05-07 2006-07-04 Wisconsin Alumni Research Foundation Image locking system for DNA micro-array synthesis
ATE474936T1 (en) 2004-06-09 2010-08-15 Wisconsin Alumni Res Found RAPID SYNTHESIS OF OLIGONUCLEOTIDES
EP1766078B1 (en) * 2004-06-18 2012-05-30 Nimblegen Systems, Inc. Variable length probe selection
WO2006002191A1 (en) * 2004-06-21 2006-01-05 Nimblegen Systems, Inc. Probe optimization methods
US7588892B2 (en) 2004-07-19 2009-09-15 Entelos, Inc. Reagent sets and gene signatures for renal tubule injury
EP1781786B1 (en) * 2004-08-27 2011-01-26 Wisconsin Alumni Research Foundation Method of error reduction in nucleic acid populations
US20060066842A1 (en) * 2004-09-30 2006-03-30 Saunders Winston A Wafer inspection with a customized reflective optical channel component
US7560417B2 (en) * 2005-01-13 2009-07-14 Wisconsin Alumni Research Foundation Method and apparatus for parallel synthesis of chain molecules such as DNA
ATE359500T1 (en) * 2005-01-18 2007-05-15 Hoffmann La Roche FLUORESCENCE IMAGING USING TELECENTRICITY
EP1859376B1 (en) * 2005-02-04 2012-05-23 Nimblegen Systems, Inc. Optimized probe selection method
US7722824B2 (en) * 2005-08-02 2010-05-25 Wisconsin Alumni Research Foundation Synthesis of arrays of oligonucleotides and other chain molecules
US20070196834A1 (en) * 2005-09-09 2007-08-23 Francesco Cerrina Method and system for the generation of large double stranded DNA fragments
US7994098B2 (en) * 2005-12-09 2011-08-09 Wisconsin Alumni Research Foundation Light directed DNA synthesis using inverse capping for error reduction
CN101553576A (en) * 2005-12-13 2009-10-07 罗氏宁博根有限公司 Method for identification and monitoring of epigenetic modifications
JP2007252249A (en) * 2006-03-22 2007-10-04 Oki Electric Ind Co Ltd Apparatus for synthesizing organic compound, light irradiation apparatus, substrate for synthesizing organic compound and method for synthesizing organic compound
US20070231823A1 (en) * 2006-03-23 2007-10-04 Mckernan Kevin J Directed enrichment of genomic DNA for high-throughput sequencing
US20070231805A1 (en) * 2006-03-31 2007-10-04 Baynes Brian M Nucleic acid assembly optimization using clamped mismatch binding proteins
US20090087840A1 (en) * 2006-05-19 2009-04-02 Codon Devices, Inc. Combined extension and ligation for nucleic acid assembly
US7759062B2 (en) 2006-06-09 2010-07-20 Third Wave Technologies, Inc. T-structure invasive cleavage assays, consistent nucleic acid dispensing, and low level target nucleic acid detection
WO2008027558A2 (en) 2006-08-31 2008-03-06 Codon Devices, Inc. Iterative nucleic acid assembly using activation of vector-encoded traits
CA2665188C (en) 2006-10-04 2014-08-19 Third Wave Technologies, Inc. Snap-back primers and detectable hairpin structures
US20080161204A1 (en) * 2006-10-06 2008-07-03 Mo-Huang Li Microwell array for parallel synthesis of chain molecules
KR100834745B1 (en) * 2006-12-20 2008-06-09 삼성전자주식회사 Oligomer probe array chip based on analysis-friendly layout, mask being used in fabrication thereof, and hybridization analysis method thereof
CA2683804A1 (en) * 2007-04-13 2008-10-23 Dana Farber Cancer Institute, Inc. Receptor tyrosine kinase profiling
ES2529790T3 (en) 2007-04-13 2015-02-25 Dana-Farber Cancer Institute, Inc. Methods of treating cancer resistant to therapeutic agents of ERBB
US7778512B2 (en) * 2007-08-28 2010-08-17 Scenterra, Inc. Light-pipe array system
EP2053132A1 (en) 2007-10-23 2009-04-29 Roche Diagnostics GmbH Enrichment and sequence analysis of geomic regions
US20090142759A1 (en) * 2007-11-29 2009-06-04 Erik Larsson qPCR array with IN SITU primer synthesis
JP2009191020A (en) * 2008-02-14 2009-08-27 Oki Electric Ind Co Ltd Apparatus for synthesizing organic compound, and method for synthesizing organic compound
US20090246788A1 (en) 2008-04-01 2009-10-01 Roche Nimblegen, Inc. Methods and Assays for Capture of Nucleic Acids
US20100056382A1 (en) * 2008-05-30 2010-03-04 Sussman Michael R Dna millichip
US20100047876A1 (en) * 2008-08-08 2010-02-25 President And Fellows Of Harvard College Hierarchical assembly of polynucleotides
WO2010025310A2 (en) 2008-08-27 2010-03-04 Westend Asset Clearinghouse Company, Llc Methods and devices for high fidelity polynucleotide synthesis
US8796011B2 (en) 2008-10-20 2014-08-05 Samsung Electronics Co., Ltd. Apparatus for fabricating and optically detecting biochip
US20100161607A1 (en) 2008-12-22 2010-06-24 Jasjit Singh System and method for analyzing genome data
US20100331204A1 (en) 2009-02-13 2010-12-30 Jeff Jeddeloh Methods and systems for enrichment of target genomic sequences
US20100292102A1 (en) * 2009-05-14 2010-11-18 Ali Nouri System and Method For Preventing Synthesis of Dangerous Biological Sequences
US9416409B2 (en) 2009-07-31 2016-08-16 Ibis Biosciences, Inc. Capture primers and capture sequence linked solid supports for molecular diagnostic tests
EP2494069B1 (en) 2009-10-30 2013-10-02 Roche Diagniostics GmbH Method for detecting balanced chromosomal aberrations in a genome
WO2011056872A2 (en) 2009-11-03 2011-05-12 Gen9, Inc. Methods and microfluidic devices for the manipulation of droplets in high fidelity polynucleotide assembly
EP3597771A1 (en) 2009-11-25 2020-01-22 Gen9, Inc. Methods and apparatuses for chip-based dna error reduction
US9216414B2 (en) 2009-11-25 2015-12-22 Gen9, Inc. Microfluidic devices and methods for gene synthesis
US9080140B2 (en) * 2009-12-01 2015-07-14 Empire Technology Development Llc Selective conformation of cell culturing support layer
WO2011085075A2 (en) 2010-01-07 2011-07-14 Gen9, Inc. Assembly of high fidelity polynucleotides
US8716467B2 (en) 2010-03-03 2014-05-06 Gen9, Inc. Methods and devices for nucleic acid synthesis
US20190300945A1 (en) 2010-04-05 2019-10-03 Prognosys Biosciences, Inc. Spatially Encoded Biological Assays
CA2794522C (en) 2010-04-05 2019-11-26 Prognosys Biosciences, Inc. Spatially encoded biological assays
US10787701B2 (en) 2010-04-05 2020-09-29 Prognosys Biosciences, Inc. Spatially encoded biological assays
WO2011128096A1 (en) 2010-04-16 2011-10-20 Roche Diagnostics Gmbh Polymorphism markers for predicting response to interleukin-6 receptor-inhibiting monoclonal antibody drug treatment
WO2011143556A1 (en) 2010-05-13 2011-11-17 Gen9, Inc. Methods for nucleotide sequencing and high fidelity polynucleotide synthesis
WO2011150168A1 (en) 2010-05-28 2011-12-01 Gen9, Inc. Methods and devices for in situ nucleic acid synthesis
US20130143214A1 (en) 2010-06-04 2013-06-06 Chronix Biomedical Prostate cancer associated circulating nucleic acid biomarkers
CN103119176A (en) 2010-06-07 2013-05-22 霍夫曼-拉罗奇有限公司 Gene expression markers for predicting response to interleukin-6 receptor-inhibiting monoclonal antibody drug treatment
EP3360963B1 (en) 2010-11-12 2019-11-06 Gen9, Inc. Methods and devices for nucleic acids synthesis
EP2637780B1 (en) 2010-11-12 2022-02-09 Gen9, Inc. Protein arrays and methods of using and making the same
GB201106254D0 (en) 2011-04-13 2011-05-25 Frisen Jonas Method and product
CN103732744A (en) 2011-06-15 2014-04-16 Gen9股份有限公司 Methods for preparative in vitro cloning
US9752176B2 (en) 2011-06-15 2017-09-05 Ginkgo Bioworks, Inc. Methods for preparative in vitro cloning
LT3594340T (en) 2011-08-26 2021-10-25 Gen9, Inc. Compositions and methods for high fidelity assembly of nucleic acids
EP2766498B1 (en) 2011-10-14 2019-06-19 President and Fellows of Harvard College Sequencing by structure assembly
PL2768985T3 (en) 2011-10-21 2019-10-31 Chronix Biomedical Colorectal cancer associated circulating nucleic acid biomarkers
WO2014163886A1 (en) 2013-03-12 2014-10-09 President And Fellows Of Harvard College Method of generating a three-dimensional nucleic acid containing matrix
WO2013123125A1 (en) 2012-02-17 2013-08-22 President And Fellows Of Harvard College Assembly of nucleic acid sequences in emulsions
FR2988093B1 (en) * 2012-03-14 2014-04-25 Alveole DEVICE FOR MICRO-STRUCTURAL GRAFTING OF PROTEINS ON A SUBSTRATE
US9150853B2 (en) 2012-03-21 2015-10-06 Gen9, Inc. Methods for screening proteins using DNA encoded chemical libraries as templates for enzyme catalysis
EP2834371B1 (en) 2012-04-05 2019-01-09 The Regents of The University of California Gene expression panel for breast cancer prognosis
EP2841601B1 (en) 2012-04-24 2019-03-06 Gen9, Inc. Methods for sorting nucleic acids and multiplexed preparative in vitro cloning
WO2013184754A2 (en) 2012-06-05 2013-12-12 President And Fellows Of Harvard College Spatial sequencing of nucleic acids using dna origami probes
TWM445196U (en) * 2012-06-06 2013-01-11 Chin-Feng Wan Maskless lithography system
TWM445178U (en) * 2012-06-25 2013-01-11 Chin-Feng Wan Microfluidic chip automatic system with optical platform
LT2864531T (en) 2012-06-25 2019-03-12 Gen9, Inc. Methods for nucleic acid assembly and high throughput sequencing
KR101743846B1 (en) 2012-07-19 2017-06-05 프레지던트 앤드 펠로우즈 오브 하바드 칼리지 Methods of storing information using nucleic acids
US9476089B2 (en) 2012-10-18 2016-10-25 President And Fellows Of Harvard College Methods of making oligonucleotide probes
EP2722105A1 (en) 2012-10-22 2014-04-23 Universität Wien Method of in situ synthesizing microarrays
US20140274749A1 (en) 2013-03-15 2014-09-18 Affymetrix, Inc. Systems and Methods for SNP Characterization and Identifying off Target Variants
US9879313B2 (en) 2013-06-25 2018-01-30 Prognosys Biosciences, Inc. Methods and systems for determining spatial patterns of biological targets in a sample
US20150141256A1 (en) * 2013-07-29 2015-05-21 Roche Nimblegen, Inc. Compositions and methods for bisulfite converted sequence capture
TWI646230B (en) 2013-08-05 2019-01-01 扭轉生物科技有限公司 Re-synthesized gene bank
US20150259743A1 (en) 2013-12-31 2015-09-17 Roche Nimblegen, Inc. Methods of assessing epigenetic regulation of genome function via dna methylation status and systems and kits therefor
CA2975855A1 (en) 2015-02-04 2016-08-11 Twist Bioscience Corporation Compositions and methods for synthetic gene assembly
CA2975852A1 (en) 2015-02-04 2016-08-11 Twist Bioscience Corporation Methods and devices for de novo oligonucleic acid assembly
EP4119677B1 (en) 2015-04-10 2023-06-28 Spatial Transcriptomics AB Spatially distinguished, multiplex nucleic acid analysis of biological specimens
US9981239B2 (en) 2015-04-21 2018-05-29 Twist Bioscience Corporation Devices and methods for oligonucleic acid library synthesis
CA2998169A1 (en) 2015-09-18 2017-03-23 Twist Bioscience Corporation Oligonucleic acid variant libraries and synthesis thereof
CN108698012A (en) 2015-09-22 2018-10-23 特韦斯特生物科学公司 Flexible substrates for nucleic acid synthesis
WO2017093246A1 (en) 2015-11-30 2017-06-08 F. Hoffmann-La Roche Ag System and method for identification of protease substrates
EP3384077A4 (en) 2015-12-01 2019-05-08 Twist Bioscience Corporation Functionalized surfaces and preparation thereof
US10184939B2 (en) 2016-02-12 2019-01-22 Roche Sequencing Solutions, Inc. Detection of neoantigens using peptide arrays
WO2018033478A1 (en) 2016-08-15 2018-02-22 F. Hoffmann-La Roche Ag Method and composition for detection of peptide cyclization using protein tags
KR102212257B1 (en) 2016-08-22 2021-02-04 트위스트 바이오사이언스 코포레이션 De novo synthesized nucleic acid library
CN109963584B (en) 2016-09-06 2024-03-15 豪夫迈·罗氏有限公司 Peptide arrays that bind MHC and methods of use thereof
US10417457B2 (en) 2016-09-21 2019-09-17 Twist Bioscience Corporation Nucleic acid based data storage
US10907274B2 (en) 2016-12-16 2021-02-02 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof
US11550939B2 (en) 2017-02-22 2023-01-10 Twist Bioscience Corporation Nucleic acid based data storage using enzymatic bioencryption
US10894959B2 (en) 2017-03-15 2021-01-19 Twist Bioscience Corporation Variant libraries of the immunological synapse and synthesis thereof
US11459618B2 (en) 2017-03-29 2022-10-04 Crown Bioscience, Inc. (Taicang) System and method for determining Cetuximab sensitivity on gastric cancer
WO2018231864A1 (en) 2017-06-12 2018-12-20 Twist Bioscience Corporation Methods for seamless nucleic acid assembly
EP3638782A4 (en) 2017-06-12 2021-03-17 Twist Bioscience Corporation Methods for seamless nucleic acid assembly
CN111566125A (en) 2017-09-11 2020-08-21 特韦斯特生物科学公司 GPCR binding proteins and synthesis thereof
KR20240024357A (en) 2017-10-20 2024-02-23 트위스트 바이오사이언스 코포레이션 Heated nanowells for polynucleotide synthesis
US10936953B2 (en) 2018-01-04 2021-03-02 Twist Bioscience Corporation DNA-based digital information storage with sidewall electrodes
EP3814497A4 (en) 2018-05-18 2022-03-02 Twist Bioscience Corporation Polynucleotides, reagents, and methods for nucleic acid hybridization
US11649485B2 (en) 2019-01-06 2023-05-16 10X Genomics, Inc. Generating capture probes for spatial analysis
US11926867B2 (en) 2019-01-06 2024-03-12 10X Genomics, Inc. Generating capture probes for spatial analysis
SG11202109283UA (en) 2019-02-26 2021-09-29 Twist Bioscience Corp Variant nucleic acid libraries for antibody optimization
US11492727B2 (en) 2019-02-26 2022-11-08 Twist Bioscience Corporation Variant nucleic acid libraries for GLP1 receptor
EP3987019A4 (en) 2019-06-21 2023-04-19 Twist Bioscience Corporation Barcode-based nucleic acid sequence assembly
US11821035B1 (en) 2020-01-29 2023-11-21 10X Genomics, Inc. Compositions and methods of making gene expression libraries
WO2023200261A1 (en) * 2022-04-13 2023-10-19 고려대학교 산학협력단 Self-alignment-based precise dna optical synthesis through image inversion

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04505763A (en) * 1989-06-07 1992-10-08 アフィメトリックス，　インコーポレイテッド Very large scale immobilized peptide synthesis
JPH05196880A (en) * 1990-06-29 1993-08-06 Texas Instr Inc <Ti> Space optical modulation device and manufacture thereof
JPH07508831A (en) * 1992-04-23 1995-09-28 マサチューセッツ・インスティチュート・オブ・テクノロジー Optical and electrical methods and devices for molecular detection
JPH0855793A (en) * 1995-08-29 1996-02-27 Nikon Corp Scanning exposure method and scanning aligner
JPH09312255A (en) * 1996-05-23 1997-12-02 Nikon Corp Aligner

Family Cites Families (43)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE2644341C2 (en) 1976-10-01 1984-08-02 Ernst Leitz Wetzlar Gmbh, 6330 Wetzlar Process and arrangements for the automatic implementation of Koehler's lighting principle
US4146329A (en) * 1977-09-14 1979-03-27 The United States Of America As Represented By The Secretary Of The Navy Autoalignment system for high power laser
JPS5534490A (en) * 1978-09-01 1980-03-11 Canon Inc Alignment device
US4571603A (en) 1981-11-03 1986-02-18 Texas Instruments Incorporated Deformable mirror electrostatic printer
US4596992A (en) 1984-08-31 1986-06-24 Texas Instruments Incorporated Linear spatial light modulator and printer
US4662746A (en) 1985-10-30 1987-05-05 Texas Instruments Incorporated Spatial light modulator and method
US5096279A (en) 1984-08-31 1992-03-17 Texas Instruments Incorporated Spatial light modulator and method
US4615595A (en) 1984-10-10 1986-10-07 Texas Instruments Incorporated Frame addressed spatial light modulator
US5202231A (en) 1987-04-01 1993-04-13 Drmanac Radoje T Method of sequencing of genomes by hybridization of oligonucleotide probes
US5525464A (en) 1987-04-01 1996-06-11 Hyseq, Inc. Method of sequencing by hybridization of oligonucleotide probes
US5028939A (en) 1988-08-23 1991-07-02 Texas Instruments Incorporated Spatial light modulator system
US5744101A (en) 1989-06-07 1998-04-28 Affymax Technologies N.V. Photolabile nucleoside protecting groups
US5424186A (en) 1989-06-07 1995-06-13 Affymax Technologies N.V. Very large scale immobilized polymer synthesis
GB8917133D0 (en) 1989-07-27 1989-09-13 Parry Davis Child safety seat
US5252743A (en) 1989-11-13 1993-10-12 Affymax Technologies N.V. Spatially-addressable immobilization of anti-ligands on surfaces
US5159172A (en) * 1990-08-07 1992-10-27 International Business Machines Corporation Optical projection system
WO1993002992A1 (en) 1991-08-07 1993-02-18 H & N Instruments, Inc. Synthesis of chain chemical compounds
US5412087A (en) 1992-04-24 1995-05-02 Affymax Technologies N.V. Spatially-addressable immobilization of oligonucleotides and other biological polymers on surfaces
DE69233087T2 (en) 1991-11-22 2003-12-24 Affymetrix Inc N D Ges D Staat Process for the production of polymer arrays
US5404783A (en) * 1992-06-10 1995-04-11 Feiten; Howard B. Method and apparatus for fully adjusting and intonating an acoustic guitar
US5324483B1 (en) 1992-10-08 1996-09-24 Warner Lambert Co Apparatus for multiple simultaneous synthesis
US5600388A (en) 1993-01-11 1997-02-04 Pinnacle Brands, Inc. Method and apparatus for creating cylindrical three dimensional picture
US5552922A (en) * 1993-04-12 1996-09-03 Corning Incorporated Optical system for projection display
US5425483A (en) * 1993-12-17 1995-06-20 Mertes; James S. Dispensing cap for vessel
US5583688A (en) 1993-12-21 1996-12-10 Texas Instruments Incorporated Multi-level digital micromirror device
US5578832A (en) 1994-09-02 1996-11-26 Affymetrix, Inc. Method and apparatus for imaging a sample on a device
US5631734A (en) 1994-02-10 1997-05-20 Affymetrix, Inc. Method and apparatus for detection of fluorescently labeled materials
US5734498A (en) 1994-05-09 1998-03-31 The Regents Of The University Of California Illuminator elements for conventional light microscopes
US5571639A (en) 1994-05-24 1996-11-05 Affymax Technologies N.V. Computer-aided engineering system for design of sequence arrays and lithographic masks
US5556752A (en) 1994-10-24 1996-09-17 Affymetrix, Inc. Surface-bound, unimolecular, double-stranded DNA
US5504614A (en) 1995-01-31 1996-04-02 Texas Instruments Incorporated Method for fabricating a DMD spatial light modulator with a hardened hinge
US5599695A (en) 1995-02-27 1997-02-04 Affymetrix, Inc. Printing molecular library arrays using deprotection agents solely in the vapor phase
US5959098A (en) 1996-04-17 1999-09-28 Affymetrix, Inc. Substrate preparation process
US5535047A (en) 1995-04-18 1996-07-09 Texas Instruments Incorporated Active yoke hidden hinge digital micromirror device
US5815310A (en) 1995-12-12 1998-09-29 Svg Lithography Systems, Inc. High numerical aperture ring field optical reduction system
US5696631A (en) 1996-02-22 1997-12-09 Anvik Corporation Unit magnification projection lens system
US5691541A (en) 1996-05-14 1997-11-25 The Regents Of The University Of California Maskless, reticle-free, lithography
US5870176A (en) 1996-06-19 1999-02-09 Sandia Corporation Maskless lithography
US5768009A (en) * 1997-04-18 1998-06-16 E-Beam Light valve target comprising electrostatically-repelled micro-mirrors
US6426184B1 (en) * 1998-02-11 2002-07-30 The Regents Of The University Of Michigan Method and apparatus for chemical and biochemical reactions using photo-generated reagents
EP2259046A3 (en) 1998-02-23 2011-11-30 Wisconsin Alumni Research Foundation Method for synthesis of arrays of dna probes
US6271957B1 (en) 1998-05-29 2001-08-07 Affymetrix, Inc. Methods involving direct write optical lithography
WO1999063385A1 (en) 1998-06-04 1999-12-09 Board Of Regents, The University Of Texas System Digital optical chemistry micromirror imager

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH04505763A (en) * 1989-06-07 1992-10-08 アフィメトリックス，　インコーポレイテッド Very large scale immobilized peptide synthesis
JPH05196880A (en) * 1990-06-29 1993-08-06 Texas Instr Inc <Ti> Space optical modulation device and manufacture thereof
JPH07508831A (en) * 1992-04-23 1995-09-28 マサチューセッツ・インスティチュート・オブ・テクノロジー Optical and electrical methods and devices for molecular detection
JPH0855793A (en) * 1995-08-29 1996-02-27 Nikon Corp Scanning exposure method and scanning aligner
JPH09312255A (en) * 1996-05-23 1997-12-02 Nikon Corp Aligner

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2023200259A1 (en) * 2022-04-13 2023-10-19 고려대학교 산학협력단 System and method for precise optical synthesis of dna

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